Poly(vinyl benzoate) nanoparticles for molecular delivery

ABSTRACT

The present invention comprises poly(vinyl benzoate) nanoparticle suspensions as molecular carriers. These nanoparticles can be formed by nanoprecipitation of poly(vinyl benzoate) in water using Pluronic F68 as surfactant, to create spherical nanostructures measuring about 200-250 nm in diameter which are stable in phosphate buffer and blood serum, and only slowly degrade in the presence of esterases. Kinetics experiments in phosphate buffer indicate that 78% of the coumarin-6 was encapsulated within the polymer matrix of the nanoparticle, and the residual 22% of coumarin-6 was surface-bound and quickly released. The nanoparticles are non-toxic in vitro towards human epithelial cells (IC 50 &gt;1000 μg/mL) and primary bovine primary aortic endothelial cells (IC 50 &gt;500 μg/mL), and exert non-observable bactericidal activity against a selection of representative test microbes (MIC&gt;250 μg/mL). Poly(vinyl benzoate) nanoparticles are suitable carriers for molecular delivery of lipophilic small molecules such as drugs pharmaceutical and imaging agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority to currentlypending U.S. Nonprovisional application Ser. No. 13/897,868 entitled“Poly(Vinyl Benzoate) Nanoparticles For Molecular Delivery”, filed May20, 2013, which is a continuation of and claims priority to prior filedInternational Application, Serial Number PCT/US2011/061429 filed Nov.18, 2011, which claims priority to U.S. Provisional Application No.61/415,003 entitled “Poly(Vinyl Benzoate) Nanoparticles For MolecularDelivery”, filed Nov. 18, 2010, the contents of which are herebyincorporated by reference into this disclosure.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant No. 0620572,awarded by the National Science Foundation. The Government has certainrights in the invention.

FIELD OF INVENTION

The present invention relates to a novel nanoparticle delivery carrierfor small molecules such as pharmaceutical and imaging agents.

BACKGROUND OF THE INVENTION

Biodegradable polymeric nanoparticles have been intensively studied as apossible way to reduce drug toxicity and degradation, while enhancingthe residence time and drug concentration at the desired site of action(K. S. Soppimath, T. M. Aminabhavi, A. R. Kulkarni, W. E. Rudzinski,Biodegradable polymeric nanoparticles as drug delivery devices, J.Control. Release 70 (1-2) (2001) 1-20; J. Panyam, V. Labhasetwar,Biodegradable nanoparticles for drug and gene delivery to cells andtissue, Adv. Drug Deliv. Rev. 55 (3) (2003) 329-347; A. Kumari, S. K.Yadav, S. C. Yadav, Biodegradable polymeric nanoparticles based drugdelivery systems, Colloids Surf. B: Biointerfaces 75 (1) (2010) 1-18).Biodegradability is an important attribute of a nanoparticle carrier forseveral reasons, including the ability to control-release the boundmolecule in a sustained, programmable way, and to provide the means forthe final removal of the carrier from the body in an innocuous form.Several biodegradable polymers have been used for this application:polylactides (PLA), co-polymers of lactic/glycolic acids (PLGA) andpoly(alkylcyanoacrylates) (PACA) are among the most widely investigatedfor drug delivery. For these systems, two degradation pathways have beenidentified: the erosion of poly(hydroxy acids) due to a main chainscission mechanism and the PACA biodegradation by side chain cleavage(V. Lenaerts, P. Couvreur, D. Christiaens-Leyh, E. Joiris, M. Roland, B.Rollman, P. Speiser, Degradation of poly(isobutyl cyanoacrylate)nanoparticles, Biomaterials 5 (2) (1984) 65-68; R. H. Müller, C. Lherm,J. Herbort, P. Couvreur, In vitro model for the degradation ofalkylcyanoacrylate nanoparticles, Biomaterials 11 (8) (1990) 590-595; J.M. Anderson, M. S. Shive, Biodegradation and biocompatibility of PLA andPLGA microspheres, Adv. Drug Deliv. Rev. 28 (1) (1997) 5-24; M. L. T.Zweers, G. H. M. Engbers, D. W. Grijpma, J. Feijen, In vitro degradationof nanoparticles prepared from polymers based on DL-lactide, glycolideand poly(ethylene oxide), J. Control. Release 100 (3) (2004) 347-356).

Release of entrapped molecules from within the matrix occurs mainly withpolymer degradation, as has been reported for poly(hydroxy acid)nanoparticles (J. M. Anderson, M. S. Shive, Biodegradation andbiocompatibility of PLA and PLGA microspheres, Adv. Drug Deliv. Rev. 28(1) (1997) 5-24). Moreover, it has been shown that PLA and PLGAnanoparticles significantly affect the viability of human granulocytes(R. H. Müller, S. Maaben, H. Weyhers, F. Specht, J. S. Lucks,Cytotoxicity of magnetite-loaded polylactide, polylactide/glycolideparticles and solid lipid nanoparticles, Int. J. Pharm. 138 (1) (1996)85-94). The PACA polymers are non-polar and more effective at entrappinghydrophobic compounds within the nanoparticles matrix, and reportedlydegrade rapidly. However, toxicity in human fibroblasts due tobyproducts resulting from degradation of the PACA backbone has beenreported (C. Lherm, R. H. Müller, F. Puisieux, P. Couvreur,Alkylcyanoacrylate drug carriers: II. Cytotoxicity of cyanoacrylatenanoparticles with different alkyl chain length, Int. J. Pharm. 84 (1)(1992) 13-22).

Polystyrene nanoparticles have also been investigated, and likewisefound to entrap lipophilic compounds to a greater extent compared topoly(hydroxy acids) nanoparticles (D. A. Norris, N. Puri, M. E. Labib,P. J. Sinko, Determining the absolute surface hydrophobicity ofmicroparticulates using thin layer wicking, J. Control. Release 59 (2)(1999) 173-185; M. C. Venier-Julienne, J. P. Benoit, Preparation,purification and morphology of polymeric nanoparticles as drug carriers,Pharm. Acta Helv. 71 (2) (1996) 121-128). This can be attributable totheir more hydrophobic nature, which may be reinforced by aromaticinteractions between pairs of benzene rings in polystyrene (J. W.Longworth, F. A. Bovey, Conformations and interactions of excitedstates. I. Model compounds for polymers, Biopolymers 4 (10) (1966)1115-1129). The higher encapsulation of polystyrene nanoparticles couldbe also attributed to stabilizing π-π interactions between its phenylgroups and heteroaromatic compounds (K. Lee, J. Hong, Nonionicadsorption of aromatic amino acids on a cation-exchange resin, React.Funct. Polym. 28 (1) (1995) 75-80).

While the prior art nanoparticles can efficiently entrap lipophiliccompounds, there are still problems with the nanoparticles degrading andproducing by-products that may affect biological structures.Additionally, some of the prior art nanoparticles degrade very rapidlythus flooding the subject with the pharmaceutical agent encapsulatedwithin the nanoparticle. A slow controlled release is not possible withthe nanoparticles of the prior art. Given the shortcomings of the priorart, what is needed is a chemically-stable biochemically-degradablenanoparticle that can efficiently entrap lipophilic substrates.

SUMMARY OF INVENTION

A long-felt but unfulfilled need exists for new chemically-stable,biochemically-degradable nanoparticles for delivery of pharmaceuticalagents such as imaging compounds, antibiotics, and anticancer drugs.

The present invention comprises poly(vinyl benzoate) nanoparticlesuspensions as molecular carriers. These nanoparticles are formed bynanoprecipitation of poly(vinyl benzoate) in water using Pluronic F68 assurfactant to create spherical nanostructures measuring about 200-250 nmin diameter. These nanoparticles are stable in phosphate buffer andblood serum, and only slowly degrade in the presence of esterases.Pluronic F68 stabilizes the nanoparticle and also protects it fromenzymatic degradation.

Up to 1.6% by weight of a lipid-soluble molecule such as coumarin-6 canbe introduced into the nanoparticle during nanoprecipitation, comparedto a water-soluble compound (5(6)-carboxyfluorescein) which gave almostno loading. Kinetics experiments in phosphate buffer indicate that 78%of the coumarin-6 was encapsulated within the polymer matrix of thenanoparticle, and the residual 22% of coumarin-6 was surface-bound andquickly released.

The nanoparticles are non-toxic in vitro towards human epithelial cells(IC₅₀>1000 μg/mL) and primary bovine primary aortic endothelial cells(IC₅₀>500 μg/mL), and exert a non-observable bactericidal activityagainst a selection of representative test microbes (MIC>250 μg/mL).Poly(vinyl benzoate) nanoparticles are suitable carriers for moleculardelivery of lipophilic small molecules such as drugs, pharmaceuticals,and imaging agents.

In an embodiment, a composition of nanoparticles is presentedcomprising: at least one nanoparticle formed from poly(vinyl benzoate)and at least one surfactant; a pharmaceutically acceptable carrier; anda lipophilic molecule. The surfactant can be a pluronic such as PluronicF68. The at least one nanoparticle may have a mean particle size ofbetween about 50 nm and about 350 nm or between about 200 nm and about250 nm. The size of the lipophilic molecule may be up to about 5% byweight. The lipophilic molecule may be an antibacterial agent or drug.

In another embodiment, an in vivo molecular carrier is presentedcomprising a poly(vinyl benzoate) nanoparticle suspension. Thenanoparticle suspension may be formed using Pluronic F68 as asurfactant. The molecular carrier may encapsulate a lipophilic smallmolecule.

In a further embodiment, a method of generating a small molecular drugcarrier is presented comprising: nanoprecipitation of a polymer, furthercomprising: adding at least one surfactant to water, where the at leastone surfactant is pluronic F68; dissolving or diluting poly(vinylbenzoate) in acetone; dissolving a predetermined quantity oflipid-soluble molecule in the acetone; adding the poly(vinylbenzoate)-acetone solution to the water to form a mixture of pluronicF68 and poly(vinyl benzoate); stirring the mixture of pluronic F68 andpoly(vinyl benzoate); and evaporating the acetone from the mixture ofpluronic F68 and poly(vinyl benzoate) to leave a suspension of polyvinylbenzoate nanoparticles.

The poly(vinyl benzoate) can be added at about 0.5% (w/v) to theacetone. The Pluronic F68 can be added at about 0.5% w/v. The acetonecan be evaporated overnight at room temperature.

The polyvinyl benzoate nanoparticles can be washed by: centrifuging thesuspension of polyvinyl benzoate nanoparticles at 10,000 rpm for 10minutes; resuspending the pellet formed from the centrifugation indistilled water; and sonicating the pellet for 1 minute to disperse anyaggregates.

The nanoparticles may be frozen at −70° C.; and lyophilized for 24hours.

Another embodiment of the present invention is a method of delivering asmall molecule to a subject comprising: administering a therapeuticallyeffective amount of a nanoparticle suspension to a subject in needthereof wherein the nanoparticle suspension is formed of poly(vinylbenzoate) and Pluronic F68. The small molecule may be lipophilic.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 is a diagram of poly(vinyl benzoate) and its hydrolysis products.

FIG. 2 is a diagram showing test fluorophores used.

FIG. 3 shows SEM micrographs of poly(vinyl benzoate) nanoparticlesprepared with Pluronic-F68 (image A) and without Pluronic-F68 (image B).SEM micrographs of Pluronic-F68-coated poly(vinyl benzoate)nanoparticles loaded with coumarin-6 (image C) and5(6)-carboxyfluorescein (image D).

FIG. 4 is a graph showing the in vitro release profile of coumarin-6from poly(vinyl benzoate) nanoparticles stabilized by Pluronic-F68.Incubation was maintained during 96 hours in PBS (pH 7.4, 10 mM) with0.1% (w/v) Tween 80 at 37° C. Data represent average value oftriplicates ±S.D.

FIG. 5 is a graph showing the percentage of benzoic acid release frompoly(vinyl benzoate) nanoparticles prepared with versus withoutPluronic-F68. Incubation was maintained at 37° C. for 32 days in 10 mMPBS (pH 7.4) containing esterase (100 U/mL), with replacement of mediumevery 24 hours. Data represent average values of three experiments ±S.D.

FIG. 6 is a chemical diagram showing the postulated in vivo metabolismof poly(vinyl benzoate).

FIG. 7 is a SEM micrograph of poly(vinyl benzoate) nanoparticles after 8days in rat serum at 37° C.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a parthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that there are other embodiments by which the invention maybe practiced. It is to be understood that other embodiments may beutilized and structural changes may be made without departing from thescope of the invention.

All numerical designations, such as pH, temperature, time,concentration, and molecular weight, including ranges, areapproximations which are varied up or down by increments of 1.0 or 0.1,as appropriate. It is to be understood, even if it is not alwaysexplicitly stated that all numerical designations are preceded by theterm “about”. It is also to be understood, even if it is not alwaysexplicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art and can besubstituted for the reagents explicitly stated herein.

Concentrations, amounts, solubilities, and other numerical data may beexpressed or presented herein in a range format. It is to be understoodthat such a range format is used merely for convenience and brevity andthus should be interpreted flexibly to include not only the numericalvalues explicitly recited as the limits of the range, but also toinclude all the individual numerical values or sub-ranges encompassedwithin that range as if each numerical value and sub-range is explicitlyrecited. As an illustration, a numerical range of “about 1 to about 5”should be interpreted to include not only the explicitly recited valuesof about 1 to about 5, but also include the individual values andsub-ranges within the indicated range, to the tenth of the unit. Thus,included in this numerical range are individual values such as 2, 3, and4 and sub-ranges such as from 1-3, from 2-4 and from 3-5, etc. This sameprinciple applies to ranges reciting only one numerical value.Furthermore, such an interpretation should apply regardless of the rangeor the characteristics being described.

The term “about” or “approximately” as used herein refers to beingwithin an acceptable error range for the particular value as determinedby one of ordinary skill in the art, which will depend in part on howthe value is measured or determined, i.e. the limitations of themeasurement system, i.e. the degree of precision required for aparticular purpose, such as a pharmaceutical formulation. For example,“about” can mean within 1 or more than 1 standard deviation, per thepractice in the art. Alternatively, “about” can mean a range of up to20%, preferably up to 10%, more preferably up to 5% and more preferablystill up to 1% of a given value. Alternatively, particularly withrespect to biological systems or processes, the term can mean within anorder of magnitude, preferably within 5-fold, and more preferably within2-fold, of a value. Where particular values are described in theapplication and claims, unless otherwise stated, the term “about”meaning within an acceptable error range for the particular value shouldbe assumed.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a nanoparticle” includes a pluralityof nanoparticles, including mixtures thereof.

The term “composition” as used herein encompasses a product comprisingthe specified ingredients in the specified amounts, as well as anyproduct which results, directly or indirectly, from combination of thespecified ingredients in the specified amounts.

The term “stabilizer” and “surfactant” are used interchangeably hereinand refers to any substance, i.e. a surface active substance, capable ofstabilizing a nanoparticle or an emulsion for preparing a nanoparticle.For example, pluronics may be used as surface active substances(surfactants).

The term “nanoparticle” as used herein refers to a particle or structurewhich is biocompatible with and sufficiently resistant to chemicaland/or physical destruction by the environment of such use so that asufficient number of the nanoparticles remain substantially intact afterdelivery to the site of application or treatment and whose size is inthe nanometer range. For the purposes of the present invention, ananoparticle typically ranges between about 1 nm to about 1000 nm,preferably between about 50 nm and about 500 nm, more preferably betweenabout 50 nm and about 350 nm, more preferably between about 200 nm andabout 250 nm. The terms “nanoparticle” and “nanostructure” are usedinterchangeably herein.

The term “nanoprecipitation” as used herein refers to the process offorming nanoparticles through solvent displacement. Innanoprecipitation, preformed polymers are converted into nanoparticlesby dissolving the polymer in a water-miscible organic solvent and addingthis solution to water containing a suitable surfactant. Generally,here, a solution of poly(vinyl benzoate) was prepared in acetone and wasintroduced into an aqueous solution containing known concentrations of asurfactant such as Pluronic F68 under magnetic stirring.

The term “lipophilic small molecule” as used herein refers to compoundswhich dissolve in lipids, fats, oils and non-polar solvents. Thelipophilic small molecule may be a pharmaceutically active agent, drug,imaging agent, therapeutic agent, diagnostic agent, compound, orcomposition. The lipophilic small molecule may comprise up to about 5%by weight of the nanoparticle composition.

The term “drug” as used herein refers to a chemical entity of varyingmolecular size, small and large, naturally occurring or synthetic, thatexhibits a therapeutic effect in animals and humans. A drug may include,but is not limited to, a therapeutic protein, peptide, antigen, or otherbiomolecule.

The term “pluronics” as used herein refers to a type of stabilizer thatmay be used during nanoparticle synthesis as an emulsion. Pluronicsprevent the adsorption of opsonins onto the particle surface(opsonization). The anti-opsonic effect of this emulsifier enhances theblood circulation time of the nanoparticles considerably, which makethem suitable for intravenous injection. In addition, pluronicsreportedly increase the uptake of pharmacologically active moleculesthrough biological membranes.

The term “degradation” as used herein refers to becoming soluble, eitherby reduction of molecular weight (as in the case of a polyester) or byconversion of the hydrophobic groups to hydrophilic groups (as in thecase of PPS).

The term “pharmaceutically active agent” as used herein refers to amolecule, a group of molecules, a complex or substance that isadministered to a subject for diagnostic, therapeutic, preventative,medical, or veterinary purposes and includes drugs and vaccines.Included are externally and internally administered topical, localizedand systemic human and animal pharmaceuticals, treatments, remedies,nutraceuticals, cosmeceuticals, biologicals, and diagnostics, includingpreparations useful in clinical and veterinary screening, prevention,prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy,surgery, monitoring, cosmetics, forensics, and the like. Thepharmaceutically active agents are preferably lipophilic forencapsulation within the nanoparticles of the present invention.

The term “polymer” as used herein refers to a relatively high molecularweight organic compound, natural or synthetic, whose structure can berepresented by a repeated small unit, the monomer. Synthetic polymersare typically formed by addition or condensation polymerization ofmonomers. The polymers used or produced in the present invention arebiodegradable. The polymer is suitable for use in the body of a subject,i.e. is biologically inert and physiologically acceptable, non-toxic,and is biodegradable in the environment of use, i.e. can be resorbed bythe body.

The term “copolymer” as used herein refers to a polymer formed from twoor more different repeating units (monomer residues).

A “pharmaceutically acceptable carrier” as used herein is defined as anyof the standard pharmaceutically acceptable carriers. The pharmaceuticalcompositions of the subject invention can be formulated according toknown methods for preparing pharmaceutically useful compositions. Thepharmaceutically acceptable carrier can include diluents, adjuvants, andvehicles, as well as carriers, and inert, non-toxic solid or liquidfillers, diluents, or encapsulating material that does not react withthe active ingredients of the invention. Examples include, but are notlimited to, phosphate buffered saline, physiological saline, water, andemulsions, such as oil/water emulsions. The carrier can be a solvent ordispersing medium containing, for example, ethanol, polyol (for example,glycerol, propylene glycol, liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. Formulations aredescribed in a number of sources that are well known and readilyavailable to those skilled in the art. For example, Remington'sPharmaceutical Sciences (Martin E W [1995] Easton Pa., Mack PublishingCompany, 19^(th) ed.) describes formulations which can be used inconnection with the subject invention.

The term “administration” as used herein refers to the delivery of acomposition of nanoparticles to an appropriate location of the subjector in vitro to which a desired effect is achieved. Routes ofadministration include, but are not limited to, intravenous,intra-arterial, intracutaneous, topical, rectal, vaginal, buccal,inhalation, ocular, and oral.

The “therapeutically effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. Atherapeutically effective amount of the nanoparticles of the presentinvention is that amount necessary to provide a therapeuticallyeffective result in vivo. The amount of nanoparticles must be effectiveto achieve a response, including but not limited to total prevention of(e.g., protection against) and to improved survival rate or more rapidrecovery, or improvement or elimination of symptoms associated with agiven disorder or other indicators as are selected as appropriatemeasures by those skilled in the art. In accordance with the presentinvention, a suitable single dose size is a dose that is capable ofpreventing or alleviating (reducing or eliminating) a symptom in apatient when administered one or more times over a suitable time period.One of skill in the art can readily determine appropriate single dosesizes for systemic administration based on the size of a mammal and theroute of administration.

The preparation and properties of poly(vinyl benzoate) nanoparticlesuspensions as molecular carriers are described for the first time.These nanoparticles can be formed by nanoprecipitation of commercialpoly(vinyl benzoate) in water using Pluronic F68 as surfactant, tocreate spherical nanostructures measuring about 200-250 nm in diameter.These nanoparticles are stable in phosphate buffer and blood serum, andonly slowly degrade in the presence of esterases. Pluronic F68stabilizes the nanoparticle and also protects it from enzymaticdegradation. Up to about 1.6% by weight of a lipid-soluble molecule suchas coumarin-6 can be introduced into the nanoparticle duringnanoprecipitation, compared to a water-soluble compound(5(6)-carboxyfluorescein) which gave almost no loading. Kineticsexperiments in phosphate buffer indicate that about 78% of thecoumarin-6 was encapsulated within the polymer matrix of thenanoparticle, and the residual about 22% of coumarin-6 was surface-boundand quickly released. The nanoparticles are non-toxic in vitro towardshuman epithelial cells (IC50N1000 μg/mL) and primary bovine aorticendothelial cells (IC50N500 μg/mL), and non-bactericidal against aselection of representative test microbes (MIC N250 μg/mL). Theseproperties suggest that the poly(vinyl benzoate) nanoparticles may besuitable carriers for molecular delivery of lipophilic small moleculessuch as pharmaceutical and imaging agents.

Given the prior art relating to nanoparticles, the inventors sought todevelop poly(vinyl benzoate) nanoparticles that, like polystyrenenanoparticles, could efficiently entrap lipophilic substrates (such asdrug molecules) (T. Hirose, K. Mizoguchi, Y. Kamiya, Gas transport inpoly(vinyl benzoate), J. Appl. Poly. Sci. 30 (1) (1985) 401-410) whilehaving the potential to bioerode by lateral chain cleavage similar tothat of PACA, but giving non-toxic by-products (polyvinyl alcohol andbenzoic acid) (FIG. 1).

In this report the methodology for the formation of poly(vinyl benzoate)nanoparticles is described, along with an investigation into theiraqueous stability and enzymatic degradability. These nanoparticles wereprepared by the nanoprecipitation method affording stable aqueoussuspensions of polymeric nanospheres in one step (H. Fessi, F. Puisieux,J. P. Devissaguet, N. Ammoury, S. Benita, Nanocapsule formation byinterfacial polymer deposition following solvent displacement, Int. J.Pharm. 55 (1) (1989) R1-4). Pluronic F68 was incorporated in the aqueousphase as a co-emulsifier in the nanofabrication process. This surfactantpresents numerous advantages as a surface coating agent fornanoparticle-based drug delivery. The hydrophilic layer of pluronicprevents the adsorption of opsonins onto the particle surface(opsonization) and therefore should prevent rapid clearance by themacrophages in the reticuloendothelial system (RES), especially in thespleen and in the liver. The anti-opsonic effect of this emulsifierenhances the blood circulation time of the nanoparticles considerably,which make them suitable for intravenous injection (D. E. Owens III, N.A. Peppas, Opsonization, biodistribution, and pharmacokinetics ofpolymeric nanoparticles, Int. J. Pharm. 307 (1) (2006) 93-102). Inaddition, pluronics reportedly increase the uptake of pharmacologicallyactive molecules through biological membranes (E. Jeon, H. D. Kim, J. S.Kim, Pluronic-grafted poly-(L)-lysine as a new synthetic gene carrier,J. Biomed. Mater. Res. 66A (4) (2003) 854-859), and have been approvedby the U.S. Food and Drug Administration for medical applications.

In order to analyze the nanoparticulate system for potential applicationin sustained drug release, the particle size and their surface chargeproperties were measured by dynamic light scattering and their surfacemorphology was characterized by scanning electron microscopy.Fluorescent probes, coumarin-6 and 5(6)-carboxyfluorescein (FIG. 2), ashydrophobic and hydrophilic drug models, respectively, were also used toencapsulate inside the poly(vinyl benzoate) matrix. The drug content andloading efficiency for each compound was then determined. The mechanismof biodegradation was further studied by enzymatic degradation and thereleased benzoic acid was used as the marker of erosion. Following this,the in vitro stability of the nanoparticles in serum was assayed toevaluate their potential for systemic applications.

Materials and Methods

Poly(vinyl benzoate) (average molecular weight 20,000-70,000) wasobtained from Monomer-Polymer and Dajac Labs, Inc. (Feasturville, Pa.).Acetone, dimethyl sulfoxide (DMSO), methanol, dichloromethane, ethanol,phosphate buffered saline (PBS), coumarin-6, 5(6)-carboxyfluorescein,benzoic acid, vinyl benzoate, Pluronic F68, Tween 80, rat blood serumand esterase from porcine liver (E.C. 232-77-7) were purchased fromSigma-Aldrich (St. Louis, Mo.). Trifluoroacetic acid (TFA), acetonitrileand water for chromatographic analysis were HPLC grade fromSigma-Aldrich as well. Potassium hydroxide was procured from FisherScientific (Fair Lawn, N.J.) and pig liver esterase was supplied by LeeBiosolutions, Inc. (St. Louis, Mo.).

The nanoparticles were prepared by the nanoprecipitation method usingsolvent displacement as reported earlier (H. Fessi, F. Puisieux, J. P.Devissaguet, N. Ammoury, S. Benita, Nanocapsule formation by interfacialpolymer deposition following solvent displacement, Int. J. Pharm. 55 (1)(1989) R1-4). Briefly, a solution of poly(vinyl benzoate) was preparedin acetone and was introduced into an aqueous solution containing knownconcentrations of Pluronic F68 under magnetic stirring. The rate ofaddition of organic phase to aqueous phase, volume ratios and thestirring speed were optimized to ensure batch-to-batch reproducibility.Typically, a 0.5% (w/v) solution of poly(vinyl benzoate) in 20 mL ofacetone was introduced into an aqueous solution of Pluronic F68 (40 mlat 0.5% w/v). The rate of addition was controlled through a syringe pumpat about 1 mL/min and stirring was maintained at a speed thatfacilitated formation of a vortex in the solution. The acetone wasevaporated overnight under stirring at room temperature to leave asuspension of polyvinyl benzoate nanoparticles. The suspension ofnanoparticles was centrifuged at 10,000 rpm for 10 minutes (EppendorfCentrifuge 5424), and the resulting pellet was resuspended in distilledwater and sonicated for 1 minute to disperse any aggregates.Centrifugation was repeated two more times at 10,000 rpm to remove theexcess Pluronic F68. After the final centrifugation, the nanoparticleswere resuspended in 10 mL of distilled water and sonicated for 2minutes. The suspension was then frozen at −70° C. and subsequentlylyophilized for 24 hours. The lyophilized nanoparticles were storeddesiccated as a dried white powder at room temperature. Drug-loadednanoparticles were prepared by pre-dissolving a known quantity(typically about 2 mg) of coumarin-6 or 5(6)-carboxyfluorescein inacetone containing the poly(vinyl benzoate) before introduction intoaqueous medium.

To determine the physical properties of the polyvinyl benzoatenanoparticles, freeze-dried samples of the nanoparticles were dispersedin deionized water and sonicated for 5 minutes. Measurements were madeby dynamic laser light scattering (DLS) using a Malvern Nano ZSinstrument. Analysis was performed in triplicate and results areexpressed as the mean volumetric diameter. Zeta potential measurementswere done by micro-electrophoresis on the same instrument. For eachsample, the average of twelve determinations was reported.

Surface morphology of the polyvinyl benzoate nanoparticles was observedby scanning electron microscopy on a Hitachi S800 SEM instrument.Samples were prepared by placing a drop of the re-dispersednanoparticles (˜10 μg/mL) on a silicon wafer and allowing the water toevaporate at room temperature for approximately 12 hours.

To determine the entrapment content and loading efficiency, nanoparticlesamples loaded with coumarin-6 or 5(6)-carboxyfluorescein were preparedas follows: 1 mg of polyvinyl benzoate nanoparticle was dissolved in 1mL of dichloromethane and this mixture was stirred for 5 hours to swellthe nanoparticles and release the entrapped molecule (fluorophore). Thissolution was left for evaporation and the residue was then solubilizedin 1 ml of acetonitrile. High performance liquid chromatography (HPLC)was done on a Shimadzu Prominence system with a reverse-phase Shimadzucolumn (C18, 0.46×5 cm). Samples were eluted using a gradient from 100%of a 10 mM PBS solution (pH 7.4) to 100% of acetonitrile in 10 minutesat a flow rate of 1 mL/minute. The detection was performed using aShimadzu SPD-20A UV-visible detector at 444 nm (coumarin-6) or at 492 nm(5(6)-carboxyfluorescein). The concentration of the fluorescent probewas determined by comparison to known concentrations of each referencecompound (using standardized curves). The amount of entrapment ofcoumarin-6 or 5(6)-carboxyfluoroscein in the nanoparticles and theiroverall loading efficiencies were calculated as follows:

Entrapment content=mass of fluorescent probe in nanoparticles/mass ofrecovered nanoparticles×100

Loading efficiency=mass of fluorescent probe in nanoparticles/mass offluorescent probe used in the formulation×100

In Vitro Release Study

The profile for release of the fluorophore in each sample was obtainedby dispersing 2 mg of coumarin-6 or 5(6)-carboxyfluorescein-loadednanoparticles in 1 mL of PBS (pH 7.4) containing 0.1% (w/v) of Tween 80in a centrifuge tube. The suspensions were stirred magnetically at 150rpm for 96 hours with a constant temperature of 37° C. Tween 80 wasadded to the suspension to ensure sink conditions as reported elsewhere(Y. Dong, S.-S. Feng, Poly(D, L-lactide-co-glycolide) (PLGA)nanoparticles prepared by high pressure homogenization for paclitaxelchemotherapy, Int. J. Pharm. 342 (1-2) (2007) 208-214). At designatedtime intervals, the samples were centrifuged at 10,000 rpm for 5minutes, the supernatant was withdrawn and the same amount of freshmedium was added. The percentage of fluorescent probe released into thesupernatant was determined by HPLC as previously described in 2.5, andthe amount of coumarin-6 released was then correlated to the amountentrapped before and after in vitro release.

Nanoparticle Polymer Degradation Experiments in Presence of Esterases

To study potential degradation properties of the nanoparticle samples, 1mg of purified, lyophilized poly(vinyl benzoate) nanoparticles(unloaded) was reconstituted in 1 mL of PBS (pH 7.4) containing 100 U/mLof pig liver esterase. Three identical samples at three differentconcentrations (100 μg/mL, 500 μg/mL, 1 mg/mL) were incubated at 37° C.for 32 days. Every 24 hours, each batch was centrifuged to separate thesupernatant from the nanoparticles and fresh medium containing 100 U/mLof pig liver esterase was added. The enzymatic cleavage of the esterbonds of the poly(vinyl benzoate) backbone causes the release of benzoicacid and polyvinyl alcohol, which was followed and the data plotted inFIG. 5. The UV-detectable benzoic acid was used as an indicator ofbioerosion and its concentration was determined by HPLC. For thischromatographic experiment, the UV detection was performed at 235 nm andthe elution was done with a gradient from 100% of 0.5% (v/v) aqueous TFAsolution to 100% of acetonitrile. The percentage of degradation isexpressed as a percentage of benzoic acid release. The theoretical fullrelease of benzoic acid was determined by total erosion of 1 mg/mL ofpoly(vinyl benzoate) nanoparticles in 1 N potassium hydroxide inmethanol. Benzoic acid concentration was established by using acalibration curve.

The degradation of the poly(vinyl benzoate) in the presence of pig liveresterase was also investigated as a control. The procedure used was asdescribed above and the experiment was extended to 14 days. It wasobserved that after two weeks in the presence of the esterase, nonotable difference of this sample compared to the poly(vinyl benzoate)nanoparticles made without Pluronic F68 surfactant.

Stability of the Poly(Vinyl Benzoate) Nanoparticles in Blood Serum

To evaluate their stability and simulate their fate in the case ofintravenous administration, lyophilized nanoparticles made with PluronicF68 were suspended in deionized water and then added to rat blood serumat a final concentration of 1 mg/mL in a water/serum ratio of 1/4 (v/v).Three suspensions were kept at 37° C. with magnetic stirring. After 1, 3and 8 days the nanoparticle samples were washed twice with deionizedwater by centrifugation at 10,000 rpm for 5 minutes. The remainingpellets were submitted to SEM analysis.

Microbiological Assays

Each of the nanoparticle samples were examined for antimicrobialactivity by testing against Staphylococcus aureus (ATCC 25923) and E.coli K12 (ATCC 23590) by Kirby-Bauer antimicrobial susceptibilitytesting on agar plates and by determination of minimum inhibitoryconcentration values by agar serial dilution. These procedures wereadapted directly from NCCLS protocols (National Committee for ClinicalLaboratory Standards, Methods for dilution antimicrobial susceptibilitytests for bacteria that grow aerobically-sixth edition (2003): ApprovedStandard M7-A6, 15, NCCLS, Wayne, Pa., USA).

Kirby-Bauer Antimicrobial Susceptibility Testing:

From a freezer stock in tryptic soy broth (Difco Laboratories, Detroit,Mich.) and 20% glycerol, a culture of each microorganism was grown ontryptic soy agar (TSA) plates (Becton-Dickinson Laboratories,Cockeysville, Md.) at 37° C. for 24 hours. A 10⁸ suspension was thenmade in sterile phosphate buffered saline (pH 7.4) and swabbed acrossfresh TSA plates. Four equidistantly-spaced circular wells (6 mm indiameter) were cut into the inoculated plates and 20, 50, or 100 μLvolumes of a 1 mg/mL stock solution of the test compound in DMSO waspipetted into the wells. 10 μL of a 1 mg/mL solution of penicillin G insterile phosphate buffered saline (PBS) was added to the fourth well (asa control). The plates were covered and then incubated for 24 hours at37° C. Antimicrobial susceptibilities were determined by measuring thediameter (in mm) of any cleared circular zones of growth inhibitionappearing around each well.

Determination of Minimum Inhibitory Concentration (MIC) by Agar SerialDilution Assays:

Sample tests were performed in 24-well plates (Costar 3524, Cambridge,Mass.) by adding known concentrations of the nanoparticle suspensions toMueller-Hinton II agar (Becton-Dickinson Laboratories, Cockeysville,Md.) for a total volume of 1 mL in each well. The final concentrationsof solid nanoparticles used in each line of wells were 256, 128, 64, 32,16, 8, and 4 μg/mL. Following the preparation of the well plates, themedia was allowed to solidify at room temperature for 24 hours beforeincubation. From a 24-hour culture of each microorganism on TSA plates,the test bacterial strains were grown overnight in 5 mL of tryptic soybroth at 37° C. to 10⁷ CFU/mL as monitored by optical densitymeasurement at 600 nm on a Bio-Tek Biomate 3 spectrophotometer. 1 μL ofeach culture was then applied to the appropriate well of agar andincubated at 37° C. overnight. After 24 h, the wells were examined forgrowth. The MIC value is defined as the lowest concentration (in μg/mL)where there was no visible growth of bacteria in the wells.

In Vitro Cytotoxicity Testing In Vitro Testing Against Epithelial Cells:

Human epithelial cells (HaCaT) from Lonza Walkersville, Inc were grownin culture medium (Dulbecco's Modified Eagle Medium (DMEM) containing10% (v/v) fetal bovine serum (FBS) and 50 □g/mL gentamycin) at 37° C.with a 5% CO₂ atmosphere for several days until cells were confluent.The cells were harvested and re-suspended in DMEM containing 10% (v/v)fetal bovine serum (FBS) and 0.1% (w/v) gentamycin. The cells werecounted using a hemocytometer, the total number of cells was determinedand the cells were seeded into 96-well plates at 50,000 cells per well.Each well contained 150 DMEM with 10% (v/v) FBS and 0.1% (w/v)gentamycin. Cells were allowed to grow for 24 hours prior to treatmentwith the nanoparticles suspensions. Testing of each dispersion atdifferent concentrations (1, 0.5, 0.25, 0.125 mg/mL) was performed intriplicate. The plates were then incubated at 37° C. and observed underthe microscope at various time points. After 48 hours, 15 μL of a 5mg/mL solution of 3-(4,5-dimethyl-2-thiazolyl)-2.5-diphenyltetrazoliumbromide (MTT) in phosphate buffered saline (10% of the total culturevolume) was added to each well. The plates were incubated for 4 hours toallow sufficient time for the conversion of the MTT dye (yellow liquid)to the water-insoluble formazan derivative,1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (purple solid) by themitochondrial dehydrogenases in the living cells. After incubation,purple crystals were observed and the media was removed from each wellby aspiration. The crystals were then dissolved by adding 100 μL of DMSOto each well. DMSO was also added to the wells designated as referenceblanks. Viable cell count was determined spectrophotometrically using amicroplate reader by measuring the absorbance at two discretewavelengths (595 and 630 nm). For each dispersion at each concentration,the absorbance values were averaged and the percent cell viability wasdetermined as a percentage of the average absorbance obtained from theuntreated cells.

In Vitro Testing Against Endothelial Cells:

Primary bovine aortic endothelial cells and EBM-2 cell culture mediumwere obtained from Lonza Walkersville, Inc. Cells were maintained inculture medium supplemented with 10% FBS and 100 IU penicillin/100 μg/mLstreptomycin sulfate in a humidified incubator containing 5% CO₂ at 37°C. For cytotoxicity testing, cells were trypsinized, counted,centrifuged at 120 g, and resuspended in fresh culture medium. Cellswere plated in a volume of 100 μL (2.5×10⁴ cells/well) in 96-wellflat-bottom culture dishes. After 16-18 h of incubation, 100 μL ofmedium or medium containing test samples was added to each well. Testingof each dispersion at different concentrations (1, 0.5, 0.25, 0.125μg/mL) was performed in triplicate. After 48 h, 20 μL of MTT (1.25-4mg/mL in DPBS) was added to each well, and the cells were furtherincubated for 2-3.5 h. The medium was aspirated, and the formazanproduct generated in each well was dissolved in 100 μL of DMSO. Plateswere read in a BioTek Synergy 2 SLFA plate reader set at 540 nm(background subtract at 660 nm). Cell viability was calculated as apercentage of the control sample (sample absorbance/control mediumabsorbance×100).

Formation and Characterization of Polyvinyl Benzoate) Nanoparticles

Research has recently focused on the preparation and studies ofnanoparticle-bound antibiotics for controlling the growth of certainpathogenic bacteria, including methicillin-resistant Staphylococcusaureus (MRSA) and Bacillus anthracis (E. Turos, J.-Y. Shim, Y. Wang, K.Greenhalgh, G. S. K. Reddy, S. Dickey, D. V. Lim, Antibiotic-conjugatedpolyacrylate nanoparticles: new opportunities for development ofanti-MRSA agents, Bioorg. Med. Chem. Lett. 17 (1) (2007) 53-56; E.Turos, G. S. K. Reddy, K. Greenhalgh, P. Ramaraju, S. C. Abeylath, S.Jang, D. V. Lim, Penicillin-bound polyacrylate nanoparticles: restoringthe activity of β-lactam antibiotics against MRSA, Bioorg. Med. Chem.Lett. 17 (12) (2007) 3468-3472; S. C. Abeylath. E. Turos, Glycosylatedpolyacrylate nanoparticles by emulsion polymerization, Carb. Polym. 70(1) (2007) 32-37; S. C. Abeylath, E. Turos, S. Dickey, D. V. Lim,Glyconanobiotics: novel carbohydrated nanoparticle antibiotics for MRSAand Bacillus anthracis, Bioorg. Med. Chem. 16 (5) (2008) 2412-2418; J.Garay-Jimenez, A. Young, D. Gergeres, E. Turos, Methods for purifyingand detoxifying sodium dodecyl sulfate-stabilized polyacrylatenanoparticles, Nanomed: Nanotech. Biol. Med. 4 (2) (2008) 98-105). Priorinvestigations have all centered on polyacrylate nanoparticles preparedas an aqueous emulsion by free-radical polymerization in water. Sodiumdodecyl sulfate is a typical surfactant for this, although othertensoactive agents can be used (J. Garay-Jimenez, D. Gergeres, A. Young,S. Dickey, D. V. Lim, E. Turos. Physical properties and biologicalactivity of poly(butyl acrylate-styrene) nanoparticle emulsions preparedwith conventional and polymerizable surfactants, Nanomed: Nanotech.Biol. Med. 5 (4) (2009) 443-451).

One of the goals is to develop suitable nanoparticle systems to protectand deliver antibiotic agents to sites of infection, where they can actdirectly on the microbe (K. Greenhalgh, E. Turos, In vivo studies ofpolyacrylate nanoparticle emulsions for topical and systemicapplications, Nanomed: Nanotech. Biol. Med. 5 (1) (2009) 46-54). Aprevious investigation reported on the formulation of poly(butylacrylate-styrene) nanoparticles as an antibiotic delivery system,altering the types of monomers and surfactants used to induce changes inparticle characteristics and antibacterial properties. It was desirableto extend those experiments toward the design of polymeric nanoparticlesthat could not only carry potentially chemically labile antibiotics, butalso might be able to undergo degradation to release the entrappedmolecule. Thus, the starting point was to replace the butylacrylate-styrene monomers used to make the nanoparticles by emulsionpolymerization with a different, potentially biodegradable variant, suchas vinyl benzoate. Attempts to prepare stable suspensions of poly(vinylbenzoate) under previously defined emulsion polymerization conditions(E. Turos, J.-Y. Shim, Y. Wang, K. Greenhalgh, G. S. K. Reddy, S.Dickey, D. V. Lim, Antibiotic-conjugated polyacrylate nanoparticles: newopportunities for development of anti-MRSA agents, Bioorg. Med. Chem.Lett. 17 (1) (2007) 53-56) using 3 weight % of sodium dodecyl sulfate(SDS) as an emulsifying agent, were unsuccessful. The use of PluronicF68 as the surfactant was changed, resulting in the formation ofnanoparticles with a mean diameter of 40 nm and a zeta potential ofapproximately −20 mV. Unfortunately, these nanoparticle emulsions werenot stable after purification by centrifugation or dialysis.Consequently, alternative means for forming nanoparticles were analyzedbased on the poly(vinyl benzoate) matrix, and the nanoprecipitationmethod was investigated. With this technique, preformed polymers areconverted into nanoparticles by dissolving the polymer in awater-miscible organic solvent and adding this solution to watercontaining a suitable surfactant.

Acetone was used as the solvent for the polymer, and satisfactoryresults were obtained with Pluronic F68. Nanoparticles were also formedwithout using an added surfactant in order to study the influence ofPluronic F68 on the degradation properties of the nanoparticles. Othersolvents that may be used in the present invention include, but are notlimited to, hardly water-soluble organic solvents having a low boilingpoint such as halogenated alkanes, methanol, ethanol, ethyl acetate,diethyl ether, cyclohexane, benzene, and toluene.

The physical characterization data of the poly(vinyl benzoate)nanoparticles formed in this manner are described in Table 1. The sizerange of about 200-250 nm as analyzed by dynamic light scattering with apolydispersity index of around 0.2 indicate a highly homogeneous sizedistribution of nanoparticles. Scanning electron microscope (SEM) images(FIG. 3) showed spherical nanoparticles with a uniformly smooth surfaceand confirmed the mean diameter of about 200-250 nm. In contrast,poly(vinyl benzoate) nanoparticles prepared in the absence of PluronicF68 were significantly larger in diameter, with a mean diameter of 490nm. Thus, the surfactant not only stabilizes the nanostructures inaqueous media, but also significantly reduces the overall dimensions ofthe nanoparticles.

TABLE 1 Characteristics of the PVBz nanoparticles Particle Poly- ZetaSize dispersity potential Nanoparticle formulations (nm) index (mV)Control with Pluronic F68 256.6 ± 7.9 0.192 ± 0.016 −29.2 ± 1.9 assurfactant Control without surfactant 489.3 ± 6.7 0.381 ± 0.010 −10.1 ±0.4 Coumarin-6-loaded nanoparticles 222.8 ± 3.1 0.099 ± 0.042 −56.6 ±0.5 5(6)-Carboxyfluorescein-loaded 206.2 ± 2.7 0.078 ± 0.007 −43.6 ± 0.8nanoparticles The results are expressed as the mean ± S.D.

Particle size is an important physical property of nanoparticlesdirectly affecting the cellular uptake capabilities, and ultimately,biodistribution. Cellular uptake is generally much greater fornanoparticles compared to microparticles (M. P. Desai, V. Labhasetwar,E. Walter, R. J. Levy, G. L. Amidon, The mechanism of uptake ofbiodegradable microparticles in Caco-2 cells is size dependent, Pharm.Res. 14 (11) (1997) 1568-1573). Hence, good cell penetration of thepoly(vinyl benzoate) nanoparticles is expected due to their small sizerange, and Pluronic-F68 is known to facilitate cell penetration, as wellas enhance in vivo stability and circulation lifetimes of the surfactednanoparticles (E. Jeon, H. D. Kim, J. S. Kim, Pluronic-graftedpoly-(L)-lysine as a new synthetic gene carrier, J. Biomed. Mater. Res.66A (4) (2003) 854-859).

The zeta potential values for the coated nanoparticles were under −30 mVfor each emulsion while −10 mV was obtained for the suspension withoutsurfactant. The zeta potential is a very important factor to evaluatethe stability of colloidal dispersion because particles are efficientlyresuspended when the absolute value of the zeta potential is at least 30mV due to the strong electric repulsion between particles (R. H. Müller,C. Jacobs, O. Kayser, Nanosuspensions as particulate drug formulationsin therapy: rationale for development and what we can expect for thefuture, Adv. Drug Deliv. Rev. 47 (1) (2001) 3-19). Therefore, knowledgeof the zeta potential can help to predict if the drug delivery system issuitable for intravenous administration due to the risk of nanoparticleagglomerations, which could result in microembolisms (P. Beck, J.Kreuter, R. Reszka, I. Fichtner, Influence of polybutylcyanoacrylatenanoparticles and liposomes on the efficacy and toxicity of theanticancer drug mitoxantrone in murine tumour models, J. Microencapsul.10 (1) (1993) 101-114).

In Vitro Toxicity Experiments

Previous studies evaluated the potential cytotoxicity of nanoparticleemulsions and methods to remove unwanted, cidal impurities createdduring emulsion polymerization (J. Garay-Jimenez, A. Young, D. Gergeres,E. Turos, Methods for purifying and detoxifying sodium dodecylsulfate-stabilized polyacrylate nanoparticles, Nanomed: Nanotech. Biol.Med. 4 (2) (2008) 98-105). Ideally, nanoparticles and all components ofthe media should induce no observable toxic effects on cells at or nearthe concentration levels needed for drug delivery. To ascertain thispotential for poly(vinyl benzoate) nanoprecipitates, the samples weresubjected to microbiological and mammalian toxicity assays. In vitroscreening of the suspensions against representative bacteria (S. aureus,E. coli) indicate that the samples are not cidal to the microbes, nor dothey inhibit bacterial growth on agar. Minimum inhibition concentration(MIC) values are above 256 μg/mL, which is above the upper limit of thetesting.

PACA nanoparticles have been found to have elevated cytotoxicity towardmammalian cell lines, in particular, human fibroblasts and epithelialtissue (C. Lherm, R. H. Müller, F. Puisieux, P. Couvreur,Alkylcyanoacrylate drug carriers: II. Cytotoxicity of cyanoacrylatenanoparticles with different alkyl chain length, Int. J. Pharm. 84 (1)(1992) 13-22; M. Brzoska, K. Langer, C. Coester, S. Loitsch, T. O. F.Wagner, C. V. Mallinckrodta, Incorporation of biodegradablenanoparticles into human airway epithelium cells-in vitro study of thesuitability as a vehicle for drug or gene delivery in pulmonarydiseases, Biochem. Biophys. Res. Commun. 318 (2) (2004) 562-570).Therefore, further in vitro testing was conducted on the poly(vinylbenzoate) nanoparticles using human epithelial cells (HaCaT) to assesspossible toxicity, over a range of about 125 to 1000 μg/mL. IC50 valuesfor the poly(vinyl benzoate) nanoparticles were greater than 1000 μg/mL,and even at this ultrahigh concentration, microscopic observation of thetreated cells showed no discernible differences versus those ofuntreated cells.

In vitro cytotoxicity studies were also conducted against primary bovineaortic endothelial cells, over a range of nanoparticle concentrationsranging from 2 to 1000 μg/mL. IC50 values of the poly(vinyl benzoate)nanoparticles was above 500 μg/mL. Microscopic imaging of the treatedcells showed no discernible differences after 48 hours versus those ofuntreated cells, at any of the concentrations. Poly(vinyl benzoate)nanoparticles are considered as non-cytotoxic in vitro given that 500μg/mL represents a much higher intravenous material dose than requiredfor in vivo drug delivery (J. M. Chan, L. Zhang, K. P. Yuet, G. Liao,J.-W. Rhee, R. Langer, O. C. Farokhzad, PLGA-lecithin-PEG core-shellnanoparticles for controlled drug delivery, Biomaterials 30 (8) (2009)1627-1634).

Molecular Loading and Release Properties of the Nanoparticle

Two fluorescent markers have been used to evaluate the potentialusefulness of the poly(vinyl benzoate) nanoparticles in drug delivery.Coumarin-6 and 5(6)-carboxyfluorescein were chosen to mimic hydrophobicand hydrophilic drugs respectively. Their lipophilicity has beenpreviously established with the octanol/water partition coefficient (P)method which is expressed as the logarithm base 10 (log P). The reportedlog P for coumarin-6 is −3.45 (C. Lombry, C. Bosquillon, V. Prèat, R.Vanbever, Confocal imaging of rat lungs following intratracheal deliveryof dry powders or solutions of fluorescent probes, J. Control. Release(2002) 83 (3) 331-341) and 5.43 for 5(6)-carboxyfluorescein (K.Lahnstein, T. Schmehl, U. Rüsch, M. Rieger, W. Seeger, T. Gessler,Pulmonary absorption of aerosolized fluorescent markers in the isolatedrabbit lung, Int. J. Pharm. (2008) 351 (1-2) 158-164).

It was observed that nanoprecipitation of 100 mg of poly(vinyl benzoate)in the presence of Pluronic F68 and an initial amount of 2 mg ofcoumarin-6 occurred with a substantial encapsulation efficiency (78%)corresponding to a drug loading content of 1.56% of the dry polymerweight. Up to about 5% by weight of a drug may be encapsulated withinthe nanoparticle. FIG. 4 shows the in vitro release profile ofcoumarin-6 in PBS at 37° C. from fluorescently-labelled nanoparticles; aburst release of about 10% was observed in the first few hours followedby a much slower rate of release. Thus, even after 96 hours, less than15% of the entrapped coumarin-6 is released from the nanoparticle.

In contrast, 5(6)-carboxyfluorescein allowed a poor loading efficiency(0.25%) leading to a total loading amount of 0.005% of the dry polymerweight (Table 2). The limit of detection by HPLC was further quantified,corresponding to about 5% of molecular release, and thus less than 5% ofthe entrapped 5(6)-carboxyfluorescein being liberated. The low amountsof burst release of the fluorophores from the nanoparticles indicatesthat the compounds are loaded by encapsulation rather than surfacesorption. This supports the expectation that hydrophobic compounds wouldbe confined inside the matrix of hydrophobic nanoparticles rather thanassociated loosely to the ionically-charged surface (J. Panyam, V.Labhasetwar, Biodegradable nanoparticles for drug and gene delivery tocells and tissue, Adv. Drug Deliv. Rev. 55 (3) (2003) 329-347; K. Y.Win, S.-S. Feng, Effects of particle size and surface coating on thecellular uptake of polymeric nanoparticles for oral delivery ofanticancer drugs, Biomaterials 26 (15) (2005) 2713-2722).

TABLE 2 Influence of the marker lipophilicity on drug contents andencapsulation efficiency Marker loaded Initial Drug Loading PVBz Logamount contents efficiency nanoparticles P (mg) (% w/w) (% w/w)Coumarin-6 — 2 1.56 ± 0.2   78 ± 3.1 3.45 5(6)- 5.43 2  0.005 ± 0.00020.25 ± 0.08 Carboxyfluorescein The results are expressed as the mean ±S.D.

It was initially postulated that poly(vinyl benzoate) would likelybiodegrade through a mechanism similar to that of PACA, i.e, enzymatichydrolysis of the ester bond between the benzoate side chain and thepolymeric backbone, with release of benzoic acid and poly(vinyl alcohol)(FIG. 1). However, there was no a priori information availableindicating whether poly(vinyl benzoate) nanoparticles could undergoappreciable enzymatic degradation in media containing esterases, or behighly resistant to hydrolytic degradation. To investigate this, theinventors incubated the freshly-prepared poly(vinyl benzoate)nanoparticles in the presence of pig liver esterase at pH 7.4 in PBS asa model medium. The release of benzoic acid from the degradation of thepoly(vinyl benzoate) nanoparticles was followed by HPLC. For this, threeconcentrations of nanoparticles (100, 500, and 1000 μg/mL) in PBS wereincubated at 37° C. with pig liver esterase (100 U/mL). Every 24-hour,the supernatant was withdrawn and replaced with fresh medium containing100 U/mL of pig liver esterase.

The inventors observed that the highest concentration of polymerproduced the most significant amount of benzoic acid as detected byHPLC. To investigate the influence of Pluronic F68 on the enzymaticprocess, the same analysis was performed with poly(vinyl benzoate)nanoparticles prepared without surfactant. In this case, afterincubation with esterases for 7 days, 4% of the benzoic acid wasreleased as evidenced by HPLC analysis. After the same duration, assayswith pluronic-coated nanoparticles liberated only 0.5% of benzoic acid.After 32 days, it was observed that 18% of the surfactantfree-nanoparticles degraded while only 2% of the benzoic acid wasdetected for the nanoparticles with Pluronic F68 (FIG. 5). These datasuggest that it would take about 5 months for the full release ofbenzoic acid in the case of nanoparticles without surfactant and about 4years for the pluronic-coated nanoparticles, when esterase is present inthe media.

The relative difference in enzymatic degradation rates observed betweenthe stabilized versus unstabilized nanoparticles shows the propensityfor pluronics to resist enzymatic degradation. Indeed, previous studieshave shown that the surface properties of polymeric nanomaterialsgreatly influence the rate of degradation and systemic lifetimes (Z.Gan, D. Yu, Z. Zhong, Q. Liang, X. Jing, Enzymatic degradation ofpoly(ε-caprolactone)/poly(DL-lactide) blends in phosphate buffersolution, Polymer 40 (10) (1999) 2859-2862). Furthermore, the slowdegradation of the poly(vinyl benzoate) nanoparticles by esterasesindicates the difficulty for enzymatic process to take place on ahydrophobic polymer nanoparticle. Other chemical and enzymaticcomponents in a complex living system may increase metabolic rates, ashas been observed for poly(ε-caprolactone) (J. S. Chawla, M. M. Amiji,Biodegradable poly(ε-caprolactone) nanoparticles for tumor-targeteddelivery of tamoxifen, Int. J. Pharm. 249 (1) (2002) 127-138).Nevertheless, with the observation that the poly(vinyl benzoate)nanoparticles undergo (albeit sluggish) side chain scission of thebenzoate ester linkage, a metabolic pathway is proposed for thenanoparticle degradation (FIG. 6). The molecular weight of thehydrolysis by-product, poly(vinyl alcohol), should be below the renalthreshold barrier and subsequently eliminated from the body primarily byrenal excretion without metabolic modification (A. Besheer, K. Mader, S.Kaiser, J. Kressler, C. Weis, E. K. Odermatt, Tracking the urinaryexcretion of high molar mass poly(vinyl alcohol), J. Biomed. Mater. Res.82B (2) (2007) 383-389; T. Yamaoka, Y. Tabat, Y. Ikada, Comparison ofbody distribution of poly(vinyl alcohol) with other water-solublepolymers after intravenous administration, J. Pharm. Pharmacol. 47 (6)(1995) 479-486). The second byproduct, benzoic acid, undergoes metabolicconversion to a benzoyl coenzyme A (CoA) adduct, which is transamidatedwith glycine to produce hippuric acid, a compound that is exported fromthe liver to the kidney to be excreted in the urine (L. Krähenbühl, J.Reichen, C. Talos, S. Krähenbühl, Benzoic acid metabolism reflectsmitochondrial function in rats with long-term extrahepatic cholestasis,Hepatology 25 (2) (1997) 278-283).

Stability in Blood Serum

The stability of poly(vinyl benzoate) nanoparticles in serum wasevaluated to further predict their potential for intravenousadministration. Freeze-dried nanoparticles were resuspended in deionizedwater then mixed with rat serum and incubated at 37° C. Thenanoparticles are stable in serum even after 8 days, keeping theiroriginal shape and size as seen in the SEM micrograph (FIG. 7).Retaining their smooth morphological features and non-agglomerated stateshould reduce the risk of microembolism and organ infraction caused bylarge coagulates in the blood (P. Beck, J. Kreuter, R. Reszka, I.Fichtner, Influence of polybutylcyanoacrylate nanoparticles andliposomes on the efficacy and toxicity of the anticancer drugmitoxantrone in murine tumour models, J. Microencapsul. 10 (1) (1993)101-114).

CONCLUSION

Nanoprecipitation of commercial poly(vinyl benzoate) in the presence ofPluronic F68 produces nanoparticles with smooth surfaces and with a meandiameter of about 200-250 nm. The nanoparticles are non-toxic towardshuman and bacterial cell lines. A hydrophobic model drug compound,coumarin-6, was efficiently encapsulated (up to 1.56% by weight) whilethe hydrophilic fluorophore, 5(6)-carboxyfluorescein, gave low compoundentrapment. Up to 5% by weight of a hydrophobic or lipophilic drug maybe encapsulated within the nanoparticle. Nanoparticles exhibited slow,controlled release in PBS. Degradation of the nanoparticles in thepresence of esterases proceeded via side chain hydrolysis to releasebenzoic acid as an indicator of bioerosion. Pluronic F68 was found toretard hydrolytic degradation, with about 5% of benzoic acid releaseoccurring within the first 32 days, while nanoparticles withoutsurfactant were degraded approximately ten times faster. Furthermore,the poly(vinyl benzoate) nanoparticles, when protected by Pluronic F68,are stable in rat serum.

The disclosures of all publications cited above are expresslyincorporated herein by reference, each in its entirety, to the sameextent as if each were incorporated by reference individually.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall there between. Now that theinvention has been described,

What is claimed is:
 1. An in vivo molecular carrier comprising ananoparticle suspension formed from poly(vinyl benzoate) and a blockcopolymer based on ethylene oxide and propylene oxide.
 2. The in vivomolecular carrier of claim 1, wherein the in vivo molecular carrierentraps a lipophilic small molecule.
 3. The in vivo molecular carrier ofclaim 2, wherein the size of the lipophilic molecule is up to about 5%by weight.
 4. The in vivo molecular carrier of claim 2, wherein thelipophilic molecule is an antibacterial agent.
 5. The in vivo molecularcarrier of claim 2, wherein the lipophilic molecule is a drug.
 6. The invivo molecular carrier of claim 1, wherein nanoparticles in thenanoparticle suspension have a mean particle size between about 50 nmand about 350 nm.
 7. The in vivo molecular carrier of claim 1, whereinnanoparticles in the nanoparticle suspension have a mean particle sizebetween about 200 nm and about 250 nm.
 8. The in vivo molecular carrierof claim 1, wherein the in vivo molecular carrier slowly degrades inpresence of esterases.
 9. The in vivo molecular carrier of claim 1,wherein the in vivo molecular carrier is stable in blood serum andphosphate buffer.